The alarming rate at which human bacterial pathogens are becoming multidrug resistant is a grave global health concern. While antibiotic-specific pharmacokinetic and pharmacodynamic knowledge is being used to administer correct dosage of antibiotics, prodigious efforts are being made to search for novel antibacterial compounds and cellular targets, and to gain a deeper understanding of the mechanism of drug resistance. One such mechanism involves the elevated expression of multidrug resistance efflux pumps to combat the drug influx across the bacterial envelope, thereby conferring resistance to a multitude of antibiotics. A major class of such drug efflux pumps, uniformly conserved in all Gram-negative human bacterial pathogens, belongs to the Resistance-Nodulation-Division (RND) family. A constitutively expressed RND pump complex of Escherichia coli comprises of AcrA, AcrB, and TolC proteins, which when assembled, bridge the inner and outer membranes. Three- dimensional structures of all three proteins from various bacterial species have been solved. Although this has brought us closer to understanding the efflux mechanism, much remains to be learned as to precisely how drugs are captured and expelled. When coupled to the movement of protons from periplasm to the cytoplasm, AcrB, the actual pump protein of the inner membrane, has the remarkable ability to bind to structurally diverse group of antibiotics and inhibitors and transport them to the channel protein TolC in the outer membrane. AcrA, a periplasmic lipoprotein anchored to the inner membrane, completes the assembly of the tripartite efflux pump. The two aims of this application will integrate structural and modeling dat with genetic, molecular dynamics simulations, and biochemical data to create a detailed map of drug binding and translocation sites in AcrB and reveal its structure-function plasticity. Given th conservation and active role of AcrB-like multidrug efflux pumps in human pathogens, the outcome of this application will have a broad and important impact.